Located between the digestive system and body circulating system in human body, liver plays an important role in defending our body from the harmful intrusion of toxic substances and in metabolism. Since foreign substances intruded into human body first passes through the liver, liver has a high possibility to be exposed to various toxic substances other than nutrients, and thus, has a higher possibility to be injured than other internal organs.
As an internal organ with excellent restoration ability, liver can completely recover its functions in slight damages. However, if liver is continuously damaged by alcohol over ingestion, chemical substance abuse, viral hepatitis, and bile secretion suspension, not only its functions are deteriorated, but a part of liver tissues are completely damaged, and the thus damaged part cannot be completely restored, which goes through liver fibrosis and may finally advance into fatal cirrhosis. Further, liver diseases do not show any pains or subjective symptoms at the initial stage, but they are found at the terminal stage. Therefore, it is impossible to treat liver diseases at a proper stage, and thus, liver diseases show a high death rate.
Regardless of the severity of liver diseases, an effective liver-disease therapeutic has not yet been found. As for liver diseases caused by viral hepatitis, anti-virus drugs are being used, but their side effects cause serious problems. As for liver diseases caused by toxic substances recently increasing due to alcohol and environment pollution, an effective liver disease therapeutic has not yet been found. Accordingly, the development of a drug, which treats and prevents liver damage while maintaining the structure and function of liver tissue is keenly required. However, since no experimental method has been developed till now, there are many limitations in developing a liver disease therapeutic. That is, in fact, there is not enough experimental support on the drugs referred to as liver protective drugs.
However, recently, an animal model has been developed which contributed to the development of a liver disease therapeutic. In this connection, an animal model induced with carbon tetrachloride is used in order to develop a liver disease therapeutic caused by toxic substances, and an acute hepatitis model induced with D-galactosamine (hereinafter abbreviated into “D-GalN”) and lipopolysaccharide (hereinafter abbreviated into “LPS”) are used in order to develop a liver disease therapeutic caused by virus.
Especially, since the above liver damage model induced with D-GalN/LPS causes liver damage by the immune reaction which is actually proceeded in most liver diseases, it is the animal model appropriate for the treatment and prevention of liver diseases [Ken-Ichiro Kosai, Kunio Matsumoto, Hiroshi Funakoshi and Toshikazu Nakamura, Hepatocyte Growth factor Prevents Endotoxin-induced Lethal Hepatic Failure in Mice. Hepatology, 1999, 30, 151-159]. In acute hepatitis model induced with D-GalN/LPS, D-GalN inhibits RNA synthesis and protein synthesis in cells to maximize liver toxicity caused by LPS, and LPS promotes the secretion and synthesis of cytokine, nitrogen monoxide (NO) and active oxygen of the kupffer cell, which is the macrophage of liver. It has been found out that tumor necrosis factor alpha (TNF-α) induced by excessive nitrogen monoxide is a main etiological agent of septicemia or acute hepatitis. In fact, it has been found out that TNF-α causes in vivo and in vitro hepatocyte death [Michael D Josephs, F. Rena Bahjat, Kunitaro Fukuzuka, Riadh Ksontini, Carmen C. Solorzano, Carl K. Edwards III, Cynthia L. Tannahill, Sally L. D. MacKAY, Edward M. Copeland III, and Lyle L. Moldawer. Lipopolysaccharde and D-galactosamine-induced hepatic injury is mediated by TNF-a and not by Fas ligand. Am J Physiol Regulatory Integrative comp Physiol, 2000, 278, R1196-R1201]. Further, Leist considers TNF-α to be the most important factor in causing liver damage by proving that the mortality is decreased when the acute liver damage model induced with D-GalN/LPS is treated with anti-tumor necrosis factor alpha (anti-TNF-α) [Leist M. Gauntner F., Bohlinger I, Germann P G, Tiegs G, Wendal A. Murine hepatocytee apoptosis induced in vitro and in vivo by TNF-α requires transcriptional arrest. J. Immunol. 1994, 153, 1778-1788].
The cell death process is largely affected by Bcl-2 family (pro- and anti-apoptotic member) proteins, which can be exemplified by Bax protein or Bid protein [Yongge Zhao, Shuchen Li, Erin E. Childs, Diane K. Kuharsky, and Xiao-Ming Yin. Activation of Pro-death Bcl-2 Family Proteins and Mitochondria Apoptosis Pathway in Tumor Necrosis Factor-a-induced Liver Injury. J. Biol. Chem. 2001, 276, 27432-27440].
More particularly, the death process of hepatocyte activates caspase 8 by interacting with FADD or TRADD protein having a death domain, when proteins inducing apoptosis such as TNF bind to the cell receptor, TNF receptor 1. The thus activated caspase 8 cleaves Bid protein and transforms it into an activated form, tBid. The thus transformed tBid is translocated to mitochondria to cause cytchrome C release. The thus released cytchrome C activates pro-caspase 9 into caspase 9, and this caspase 9 induces the cooperative effects of lower caspases by activating caspase 3 which leads all cells to apoptosis [Xiao-Ming Yin, Bid, a critical mediator for apoptosis induced by the activation of Fas/TNF-R1 death receptors in hepatocytes. J. Mol, 2000, 78, 203-211].
Therefore, hepatocyte apoptosis generated in acute liver-injury model induced with D-GalN/LPS causes the activation of apoptosis pathway by TNF-α receptor. Accordingly, it can be proved that an extract of the stem or root of Acanthopanax koreanum activates liver protection by the said working by examining whether Acanthopanax koreanum polysaccharide inhibits TNF-α activity itself, and by proving that Acanthopanax koreanum polysaccharide inhibits the expression of the important protein activated by TNF-α.
In addition, the amount of circulating alanine aminotransferase (hereinafter abbreviated into ALT, GPT index) and aspartate aminotranasferase (hereinafter abbreviated into AST, GOT index), and the concentration of circulating tumor necrosis factor (TNF-α) are measured to determine liver protection activity in the acute liver damage model induced with D-GalN/LPS. In addition, the liver protection effect of the sample can be determined more precisely by measuring apoptosis inhibition effect of hepatocyte using the activity inhibiting hepatocyte DNA cleavage as an index, and by measuring the 24 hour survival rate of the mouse.
Recently a drug for the treatment or prevention of hepatitis by protecting the liver functions by using the said animal model is being developed. Especially, it has been reported that saponin, bupleuroside compounds (H. Maysuda et al., Bioorg. Med. Chem., 1997, 7, 2193-2198), naringin (K. Kawaguchi et al., Eur. J. Pharmacol., 1999, 368, 245-250), green tea extract(P. HE et al., J. Nutr., 2001, 131, 1560-1567), polysaccharides extracted from the seeds of Celosia argentea show activity in protecting the liver functions in the liver damage model induced with D-GalN/LPS and inhibiting the experimental animal lethality (K. Hase et al., Biol, Pharm. Bull., 1996, 19, 567-572).
In addition, there is a report on the liver protection activity of an extract of Acanthopanax senticosus [Chun-Ching Lin and Pei-Chen Huang, Phytotherapy Research, 2000, 14, 489-494]. However, no specific examples on an extract of Acanthopanax koreanum for the treatment or prevention of hepatitis or liver protection have been reported. Acanthopanax senticosus morphologically differs a lot from Acanthopanax koreanum. Acanthopanax senticosus is thickly wooded with thin long thorns on its bark and branches, and the style of the fruit is divided into 5. Further, it is mainly distributed in the alpine regions of Korea; Hokkaido, Japan; the Heilung Riverside, China; and Siberia, Russia. Acanthopanax koreanum is wooded with triangle shaped grayed-brown thorns with a large base, and the style of its fruit is divided into 2. Further, it is a Korean indigenous plant distributed in the southern part of Korea including Chejudo.
Further, Acanthopanax koreanum comprises acanthoic acid; pimara-9(11)-dien-19 oic acid as its main component, whereas, Acanthopanax senticosus does not comprise such component [Young H. Kim and Bo S. Chung J. Nat. Prod. 1988, 51 1080-1083].
Therefore, in connection with liver diseases caused by hepatitis virus and toxic substances, inventors have devoted themselves in developing a liver disease therapeutic with less side effects, and based on the working model of the acute liver-injury induced with D-GalN/LPS the present invention has been completed by proving an accurate experimental method and results that an extract from the root or stem of Acanthopanax koreanum is effective on the treatment and prevention of liver damage while maintaining the structure and function of the liver tissues.